Green synthesis of copper oxide nanoparticles using walnut shell and their size dependent anticancer effects on breast and colorectal cancer cell lines

Metal oxide nanoparticles(NPs) contain unique properties which have made them attractive agents in cancer treatment. The CuO nanoparticles were green synthesized using walnut shell powder in different calcination temperatures (400°, 500°, 700°, and 900 °C). The CuO nanoparticles are characterized by FTIR, XRD, BET, SEM and DLS analyses. SEM and DLS analyses showed that by increasing the required calcination temperature for synthesizing the NPs, their size was increased. DPPH analysis displayed no significant anti-oxidative properties of the CuO NPs. The MTT analysis showed that all synthesized CuO NPs exhibited cytotoxic effects on MCF-7, HCT-116, and HEK-293 cell lines. Among the CuO NPs, the CuO-900 NPs showed the least cytotoxic effect on the HEK-293 cell line (IC50 = 330.8 µg/ml). Hoechst staining and real-time analysis suggested that the CuO-900 NPs induced apoptosis by elevation of p53 and Bax genes expression levels. Also, the CuO-900 NPs increased the Nrf-2 gene expression level in MCF-7 cells, despite the HCT-116 cells. As can be concluded from the results, the CuO-900 NPs exerted promising cytotoxic effects on breast and colon cancer cells.

attention has been recently paid to the green approach and biological method to the synthesis of nanoparticles, chiefly copper oxide nanoparticles [16][17][18][19] .
Agricultural waste biomass particularly lignocellulosic wastes such as nut shells have increasingly attracted some attention as a low-cost renewable resource in various fields 20,21 .Walnut is always a popular fruit in the world.Based on incomplete statistics, thousands of tons of walnuts are consumed every year in the world and the shells are usually cast-off.More recently, we synthesized nanostructured magnesium oxide 22 , alumina 23 , and cerium oxide nanoparticles 24 by walnut shell.This abundantly available agricultural waste is composed of cellulose, hemicellulose, and lignin 5,25 which can act as fuel or sacrificial templates in preparing metal oxide nanostructures.In this protocol, walnut shell grind was mixed with aqueous solutions of metal nitrate in diverse ratios without using any toxic chemicals.Stirring followed by evaporation and calcination of paste, resulting in desired nanostructures.In this paper, we present similar process for the preparation of CuO nanoparticles with different sizes by using walnut shell powder.The aim of this study is the green synthesis of different dimensions of CuO NPs to investigate their anticancer effects.

General remarks
Copper(II) nitrate hexahydrate, Cu(NO 3 ) 2 .6H 2 O, from Merck and used without further purification.The walnut shell from a local walnut tree in Urmia (Iran) was crushed using a high-speed rotary cutting mill.X-ray diffraction patterns of the obtained materials were recorded at room temperature on Shimadzu XRD-6000 diffractometer with CuKα irradiation.The morphology of materials was observed by Hitachi S-4100 FESEM instrument (Japan).Also, BET analysis was performed by Belsorp-mini II-BEL, Inc. analyzer at 77 K. Fourier transform infrared (FT-IR) spectra were attained by a Bruker Vector 22 FT-IR spectrophotometer under ambient conditions in a KBr/Nujol mull in the range of 400-4000 cm −1 .

Copper oxide nanoparticles preparation
Walnuts were purchased from local farmers and then the walnut shells separated from the kernel.Next, the walnut shells were grinded and the walnut shell powder was prepared.Copper oxide NPs was synthesized in the presence of walnut shell powder 30 g and copper(II) nitrate hexahydrate 6.65 g in 50 mL of deionized water (Millipore, Milli-Q grade).After 4 h stirring, the water was evaporated by a rotary evaporator under low pressure.The resulting paste was subsequently calcined at 400 °C for 4 h (at a heating rate of 10 °C/min) under open-air conditions to give a black solid (CuO-400).For comparison, CuO-500, CuO-700, and CuO-900 NPs were prepared at 500, 700, and 900 °C, respectively.A schematic diagram of Copper oxide NP preparation is shown in Fig. 1.
For evaluation the cytotoxic effect of the CuO nanoparticles produced at different temperatures (400 °C, 500 °C, 700 °C, and 900 °C) as well as CuO bulk, 5 × 10 3 cells were seeded in 96 well plate and exposed to different concentrations of the nanoparticles(20,40, 80 µg/ml prepared in distilled water) for 72 h at 37 °C.After that, 10 µl of MTT dye solution (5mg/ml, Sigma) was added to each well and the plates were incubated at 37 °C in dark.
Next, after removing each well medium, 100µl DMSO (Dimethyl sulfoxide, Merck) was poured into each well to dissolve the formazan crystals.Finally, the wells absorbance was read at 570 nm and based on the control wells absorbance, the treated cells viability were calculated and the plots drawn by GraphPad prism Ver.9.0.0.

Hoechst staining to monitor apoptosis
In the apoptosis process, cells undergo some morphological alterations including nucleus condensation and fragmentation as well as apoptotic body formation 27 .The Hoechst33342 dye can bind to DNA and monitor the cell nucleus shape.First 2 × 10 5 cells(HCT-116 and MCF-7 cell line) were seeded in each well of 24 well plate, then treated with 80µg/ml concentrations of the CuO nanoparticles produced at 400, 500, 700, and 900 °C as well as CuO bulk for 72 h.In order to Hoechst staining, the cells first washed once by PBS(phosphate buffered saline)and trysinized that followed by fixing in ice cold methanol(Merck) at − 20 °C for 30 min.Thereafter, the cells were centrifuged and the supernatant was discarded and the cell pellets were twice washed in PBS.In the next step, the cells stained by Hoechst 33,342 satin solution(Sigma, 1mg/ml) for 30 min out of light.Finally, the cells were transferred on glass coverslips and photographed by a fluorescent microscope (Zeiss) by a DAPI filter.

Q-PCR to analyze apoptotic and antioxidant genes expression
To evaluate the apoptotic and antioxidant genes expression by real-time PCR method, first, 2 × 10 6 MCF-7 and HCT-116 cells were seeded in each well of 6-well plate and then exposed to 80 µg/ml concentration of CuO-900 NPs.After that, the cells were trypsinized and centrifuged to obtain the cells pellets.Next, the cells RNAs were extracted by RNXplus kit(Sinaclon, Iran) based on manufacture's protocol.After determining the RNAs concentrations by a Nanodrop (Biotek), the cDNA synthesis was performed using 1000 ng extracted RNA by cDNA synthesis kit(Parstus, Iran).Next, the samples cDNA were subjected to real-time PCR (Applied Biosystems) in the presence of Bax, Bcl-2, p53, Nrf-2, superoxide dismutase, catalase and Beta-action(as loading control) primers using sybergreen mastermix(Ampliqon) as the following steps: Denaturation at 94 °C for 5 min, 40 cycles of Denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, elongation at 72 °C for 30 s.
All the primers sequence was designed by using Oligo 7 software, the primers sequences have been presented at Table 1.

Statistical analysis
The experiments were repeated at least three times (n = 3) and the obtained data were analyzed via one or two-way ANOVAs using the Tukey post hoc test by GraphPad prism Ver.9.0.0 software.The p-value < 0.05, 0.01, 0.001, and 0.0001 were considered as significant difference between the control and treated groups.

CuO NPs were synthesized in different sizes
The FTIR spectrums of CuO nanoparticles obtained by process described above are presented in Fig. 2. The FTIR spectrum shows peaks at 400-600 cm -1 , which can be assigned to the vibrations of Cu-O bonds.Also, peaks at around 1400 cm −1 are attributed to carbon residue that an increased calcination temperature was followed by removal of carbon impurities and shortening of these peaks.X-ray diffraction(XRD) analysis of synthesized different-sized CuO NPs were shown in Fig. 3.The XRD pattern matched with the monoclinic structure of CuO crystal(JCPDS card 80-1917).An intense diffraction peak at 38.77° was detected corresponding to the lattice plane (111), in addition to this, some low-intensity peaks at 35.74° (002) and 48.94° (202), were also detected.The effect of temperature was also studied on crystal growth, showing the small changes in the sharpness of peaks and growth of ( 111) and (002) planes in CuO-400, Fig. 2. FT-IR analysis.In CuO-900 NPs the peak of wavenumber 1400 cm −1 has been disappeared.=X-ray wavelength (0.15418 nm), β = FWHM, D = crystallite size, θ = Bragg's angle.
Additionally, the specific surface area and particle sizes of CuO NPs can be determined using the BET surface area (Table 1).The method works based on nitrogen gas adsorption at a constant temperature.This analysis indicated the average thermodynamic size of CuO-400 to be 71 nm.It has been observed that with the increase in temperature of calcination the average size increase to 127, 201, and 295 nm for CuO-500, CuO-700, and CuO-900, respectively.An increase in calcination temperature results in growing of crystals (coarsening or Ostwald ripening) and hence higher particle as well as crystallite sizes.
The BET results were further supported by FESEM images and a histogram of the particle size distribution (Fig. 4 and Table 2).From the FESEM images, it is clearly evident that with raising temperature, the www.nature.com/scientificreports/average size and size distribution of nanoparticles also increases, and the interesting thing is that rod-like nanoparticles are also seen in CuO-900.It is also evident that nanoparticles at higher temperatures are more geometrically regular than nanoparticles prepared at lower temperatures.By carefully looking at the histograms, it can be seen that the highest frequency of nanoparticles in all 4 samples is related to nanoparticles under 100 nm, and in sample CuO-400, it is related to nanoparticles under 50 nm, but with increasing temperature, the size distribution of nanoparticles has expanded to larger values.As can be seen, the size of the particles obtained from DLS analysis is much larger than the size obtained using the FESEM histogram (Fig. 5).This difference is due to the principle that the DLS technique provides the hydrodynamic diameter of agglomerated particles rather than the real size of nanoparticles 28 .An increase in calcination temperature results in larger size of agglomerated particles and bimodal distribution.
The proposed mechanism of the CuO nanoparticle formation is pictured in Fig. 6.It is possible that the Cu 2+ ions were dispersed on the walnut shell containing cellulose, hemicellulose, and lignin via coordination with their alkoxy, hydroxyl, and other oxygenated groups.Therefore, walnut shell act as an adsorbent for the copper precursors.After adoption and dispersion of the Cu 2+ on the walnut shell and calcination, the template was removed (walnut shell as sacrified template or fuel) by transformation of CO, CO 2 , and H 2 O (Fig. 6).Simultaneously, due to temperature increase CuO is formed with removal of template.The product should be nanostructured due to two reasons including 1-use of the template and 2-release of gases which increase the surface area (Fig. 6).

CuO nanoparticles especially CuO-900 NPs exerted anticancer effects
To determine cytotoxic effects of the synthesized CuO nanoparticles, the MCF-7, HCT-116, and HEK-293 cell lines were treated with different concentrations(20, 40, and 80 µg/ml) of the CuO nanoparticles(CuO-400, CuO-500, CuO-700, and CuO-900) as well as CuO bulk for 72 h (Figs. 8, 9, 10, 11 and 12).Because the CuO nanoparticles exhibited the most cytotoxic effects in 72 h (Figs. 8, 9, 10, 11 and 12), so, the treatment time was chosen to calculate the IC 50 values (Table 3).After analyzing the MTT data, the nanoparticles IC 50 values were calculated and summarized in Table 1.As the table showed, the nanoparticles exerted cytotoxic effects on the MCF-7, HCT-116, and HEK-293 cell lines, but the CuO-900 NPs showed lesser cytotoxic effect on HEK-293 cell line(IC 50 = 330.8µg/ml, Table 3) compared with the other CuO NPs.Accordingly, the CuO-900 nanoparticles exhibited an appropriate anticancer effect due to their lesser cytotoxic effects on the normal cell line, so, the CuO nanoparticles were chosen for further studies.As Figs. 8, 9, 10, 11 and 12 showed, the cytotoxic effects of all CuO NPs and also the CuO-bulk were in a time and dose-dependent manner.In a more recent work, Dutta et al. reported that biogenic CuO NPs derived from Erythrina variegate display cytotoxic effect on HeLa cell line(IC 50 = 48 µg/ml), but the nanoparticles showed lesser toxic effect on HEK293 cell line 29 .In another work, the CuO nanoparticles synthesized via Annona muricata extract reduced breast cancer cells proliferation 10 .Additionaly, in another work, the CuO NPs derived from Houttuynia cordata displayed cytotoxic effects on cervical cancer cells 11 .Moreover, Sankar et al. have reported that CuO NPs synthesized from Ficus religiosa extract reduced A549 cell viability in dose dependent manner 30 .It has been reported that CuO NPs exhibited dose dependent cytotoxic effect on PANC-1 cancer cell line 12 .Therefore, this work results were similar to the previous works.Moreover, our results showed that the anticancer effect of the CuO NPs is correlated to the size of the NPs.The CuO-900 NPs exerted lower cytotoxic effects on HEK-293 cell line rather than other CuO NPs.Because IC 50 value of the CuO-900 NPs on HUVEC cells was higher than it on MCF-7 and HCT-116 cells(Table 3), therefore, the CuO NPs were chosen as an appropriate anticancer agent to analyze the apoptotic and antioxidant genes expression in MCF-7 and HCT-116 cell lines.

All of the CuO nanoparticles induced apoptotic cell death
Apoptosis is a type of cell death that removes unwanted cells without damaging other cells; Therefore, this type of death is an important way for removing cancer cells 31 .Benguigui et al. reported that CuO NPs induce apoptosis in the PANC-1 cell line 12 .In another study, CuO NPs induced apoptosis in HepG2 cells 7 .In apoptotic cells, the cell nucleus undergoes some alterations, including condensation in early apoptosis and fragmentation in late apoptosis 32 .Dutta et al. showed biogenic CuO NPs induce apoptosis in HeLa cells 29 .In this work, the apoptosis inducing effects of the CuO NPs were analysed by Hoechst 33,342 staining to detect the apoptotic cells nuclei.As shown in Fig. 13, in the treated cells, the dense or fragmented nuclei were observed.In the HCT-116 cells, the most apoptotic nuclei were observed in the CuO-900 NPs treated cells, and the least apoptotic nuclei were obtained in CuO-700 treated cells (Fig. 13a).In the MCF-7 cells, the CuO bulk induced the most apoptotic nuclei.Additionally , in the cells, the most and least apoptotic nuclei were seen in CuO-500 and CuO-700 NPs treated cells, respectively (Fig. 13b).The results suggest that the cell viability reduction in the treated cells can be exerted by apoptosis induction.These data confirmed the MTT assay results.www.nature.com/scientificreports/

Effect of CuO-900 nanoparticles on apoptotic genes expression
To illuminate the role of the CuO-NPs on apoptosis induction, real-time PCR analysis was done.As Fig. 14b showed, in treated MCF-7 cells, the expression levels of p53 and Bax genes were higher than untreated cells (p53:p control vs treated < 0.0001, Bax: p control vs treated < 0.05) while the expression level of Bcl-2 was not significantly altered in compared to the untreated cells(p = 0.9994).As shown in Fig. 1a, the expression level of p53 and Bax genes was increased in HCT-116 treated cells with compared to untreated cells(p53:p control vs treated = 0.0016, Bax: www.nature.com/scientificreports/p control vs treated < 0.0001).In another hand, The expression level of Bcl-2 did not exhibit a significant change in treated cells(p = 0.999).The Bax/Bcl-2 ratio level in MCF-7 and HCT-116 cells were 1.66 and 3.5, respectively.
According to an elevation of the Bax/Bcl-2 ratio level, which is an indicator of apoptosis, the results of the realtime analysis confirmed the hoechst 33,342 staining results that indicating apoptosis occurence in CuO-900 NPs treated MCF-7 and HCT-116 cell lines.The results are similar to a recent work's results, which reported CuO NPs derived from Bacillus Coagulus elevate the Bax/Bcl2-2 level in MCF-7 and SKBR3 cell lines 33 .It was reported that DNA damage and reactive oxygen species increase the p53 gene expression 34 .Therefore, CuO-900 NPs may increase the p53 gene expression by DNA damage or ROS production, which leads to apoptosis induction.

CuO-900 NPs exhibit different effect on Nrf-2 gene expression in the MCF-7 and HCT-116 cell lines
The antioxidant activity of the CuO NPs was measured by the DPPH test.As Fig. 7 showed, the CuO NPs did not display antioxidant activity.Despite , in previous works, the green synthesized CuO NPs exhibited antioxidant activity [35][36][37] .According to the application of high temperatures in the synthesis procedure of the CuO NPs, it can be suggested that the high temperatures degrade the organic compounds which cause antioxidant activity.
To investigate the effect of CuO-900 NPs on the antioxidant genes expression profile, real-time analysis was employed.Nrf-2(nuclear factor erythroid 2-related factor-2) is a transcription factor which plays a pivotal role in the redox state of cells by overexpressing of own gene expression as well as other redox genes expression, such  as superoxide dismutase(SOD) and catalase(CAT) enzyme genes [38][39][40] .Therefore, Nrf-2 overexpression reduces the oxidative stress in cells 41 .Based on the role of the Nrf-2 , superoxide dismutase and catalase in the redox state of the cells, their gene expression level were chosen to investigate the redox genes expression analysis in the CuO-900 NPs treated cells.Tabatha et al. showed that CuO NPs activate the Nrf-2 pathway in the JB6 cells 42 .
As shown in Fig. 14b, the Nrf-2 and SOD and CAT genes expression level was increased in MCF-7 treated cells (1.9, 3.4, and 3.3 times, respectively).The effect may be caused by CuO-900 NPs mediated ROS production that

Table 2 .
Surface area, BET particle sizes and FESEM particle sizes of copper oxide nanoparticles.